High output nitric oxide (NO) synthesis has been linked to airway inflammation in asthma. NO is produced by human airway epithelial cells (HAEC) which have increased NO synthase 2 (NOS2) expression in asthma due to transcriptional activation of the gene. Our preliminary results show that interferon gamma (IFNgamma) induces, and interleukin-4 augments, NOS2 expression in HAEC in vitro through mechanisms that require physical interaction between activator protein (AP)-I and IFNgamma-activated signal transducer and activator of transcription (STAT)-I prior to DNA binding. In the context that asthma results from inflammatory processes that injure or modify airway function, excessive NO may participate in the pathogenesis of asthma through reactive nitrogen species formation and subsequent nitration of proteins which modifies their biologic functions. In support of this, nitrated proteins are increased in the asthmatic airway. Using an innovative proteomic approach, we have identified tyrosine nitration of specific proteins in lung epithelial cells after NOS2 induction, and in the human airway, e.g. Mn superoxide dismutase and catalase. Nitration of antioxidants leads to inactivation, and biologic consequence of apoptosis. Taken together, we hypothesize that NO synthesis is increased in asthma due to NOS2 gene induction by AP-1 and STAT-1 signaling mechanisms in the airway epithelial cell, and that the generation of excess NO leads to protein tyrosine nitration which modifies protein functions and contributes to airway injury/inflammation. To test this hypothesis, we will (1) define the regulation of NOS2 gene expression using HAEC in culture, and airway cells freshly obtained from asthmatic and healthy control lungs, and (2) determine the role of NO in asthma by a proteomic approach to identify nitrated proteins before and after NOS2 induction in HAEC in culture, and in clinical samples from asthmatics in comparison to patients with chronic obstructive pulmonary disease (COPD) and controls. Identification of nitrated proteins and consequences of biological tyrosine nitration will provide clear and detailed information on the role of NO in asthma. Since all the studies are performed in the physiologically relevant context of the human lung or in primary airway epithelial cells, these studies will provide a valuable comprehensive picture of the mechanisms that control NO synthesis in the human airway, and alterations in those mechanisms that lead to asthma.